High throughput genome engineering has been used to create organisms with designed genomes. See Smith, H. O., Hutchison, C. A., Pfannkoch, C. and Venter, J. C. (2003), Generating a synthetic genome by whole genome assembly: phi X174 bacteriophage from synthetic oligonucleotides, Proc. Natl. Acad. Sci. U.S.A., 100, 15440-15445 and Gibson, D. G., Glass, J. I., Lartigue, C., Noskov, V. N., Chuang, R. Y., Algire, M. A., Benders, G. A., Montague, M. G., Ma, L., Moodie, M. M. et al. (2010), Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, Science, 329, 52-56. Certain methods of genome engineering involving recombination are known. See Wang, H. H., Isaacs, F. J., Carr, P. A., Sun, Z. Z., Xu, G., Forest, C. R. and Church, G. M. (2009), Programming cells by multipleX genome engineering and accelerated evolution, Nature, 460, 894-898; U.S. Pat. No. 8,153,432; See Can, P. A., Wang, H. H., Sterling, B., Isaacs, F. J., Lajoie, M. J., Xu, G., Church, G. M. and Jacobson, J. M. (2012), Enhanced MultipleX Genome Engineering through Cooperative Oligonucleotide Co-selection. Nucleic Acids Res., 1-11; Zechner et al., Coordinated leading- and lagging-strand synthesis at the E. Coli DNA replication fork. II. Frequency of primer synthesis and efficiency of primer utilization control of Okazaki fragment size, Journal of Biological Chemistry, 267, 4045-4053 (1992). See Wang, H. H., Kim, H., Cong, L., Jeong, J., Bang, D. and Church, G. M. (2012), Genome-scale promoter engineering by coselection MAGE, Nat Meth, 9, 591-593. Such methods typically involve introducing eXogenous DNA into the genomes of dividing cells. Such methods can utilize phage λ Redβ recombinase, which binds to ssDNA oligos, protecting them from ssDNA eXonucleases, and facilitating their annealing to the lagging strand of the replication fork. See Ellis, H. M., Yu, D. G., DiTizio, T. and Court, D. L. (2001), High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides, Proc. Natl. Acad. Sci. U.S.A., 98, 6742-6746. Generating a heterogenic population has been harnessed for directed evolution of biosynthetic pathways and eXtensive cycling toward isogenic populations has been used to remove all 314 TAG stop codons in subsets across 32 E. coli strains. See Isaacs, F. J., Carr, P. A., Wang, H. H., Lajoie, M. J., Sterling, B., Kraal, L., Tolonen, A. C., Gianoulis, T. A., Goodman, D. B., Reppas, N. B. et al. (2011), Precise manipulation of chromosomes in vivo enables genome-wide codon replacement, Science, 333, 348-353.
Several approaches are known for improving introduction of eXogenous nucleic acids into the genome of a cell such as targeting oligos to the lagging strand of the replication fork, See Li, X. T., Costantino, N., Lu, L. Y., Liu, D. P., Watt, R. M., Cheah, K. S., Court, D. L. and Huang, J. D. (2003), Identification of factors influencing strand bias in oligonucleotide-mediated recombination in Escherichia coli, Nucleic Acids Res, 31, 6674-6687, evading mismatch repair using modified nucleotides, See Wang, H. H., Xu, G., Vonner, A. J. and Church, G. M. (2011), Modified bases enable high-efficiency oligonucleotide-mediated allelic replacement via mismatch repair evasion, Nucleic Acids Res, 39, 7336-7347, minimizing oligo secondary structure and optimizing homology lengths, blocking oligo degradation with 5′ phosphorothioate bonds, avoiding sequences with high degrees of off-target homology elsewhere in the genome, and removing the mismatch repair protein MutS to avoid reversion of mutated alleles. See Costantino, N. and Court, D. L. (2003), Enhanced levels of lambda Red-mediated recombinants in mismatch repair mutants, Proc Natl Acad Sci USA, 100, 15748-15753.
Okazaki Fragment (OF) size can be modulated by the frequency of OF primer synthesis by DnaG primase. Tougu et al. have reported E. coli primase variants with impaired helicase binding, resulting in less-frequent OF initiation, but normal replication fork rate, priming efficiency, and primer utilization during in vitro replication. These variants, K580A and Q576A, resulted in in vitro OFs that were approXimately 1.5- and 8-fold longer (respectively) than those initiated by wild type (wt) DnaG. See Tougu, K. and Marians, K. J. (1996), The EXtreme C Terminus of Primase Is Required for Interaction with DnaB at the Replication Fork, Journal of Biological Chemistry, 271, 21391-21397.